Traumatic brain injury (TBI) is brain damage resulting from an external mechanical force, such as blast or crashes. Our current understanding of TBI is derived mainly from in vivo studies that show measurable biological effects on cells sampled after TBI. Little is known about the primary mechanical stresses in brain cells during damaging stimuli, and how they are transduced into cellular and molecular events involved in TBI. This proposal aims to examine what stimulus properties are most critical to cell injury and how the mechanical stimulus is coupled to secondary cellular processes. We will use tissue cultured adult astrocytes in a microfluidic chamber driven by a fast pressure servo to generate time dependent fluid shear that can be made to emulate shear stress from a blast. We will directly measure the stresses in specific cytoskeletal proteins using genetically encoded fluorescent stress sensors. The research has two specific aims. Aim 1 identifies physical properties of the stimulus that lead to long term alterations. By measuring the time course of stresses in specific cytoskeletal proteins using shockwaves of various amplitude, rise time, and frequency, we will determine the parameters that lead to irreversible deformation. This irreversibility will be determined by investigating changes in anatomy and stress distribution. Aim 2 investigates the cells' response during and immediately after mechanical insult to determine how mechanical stimulus alters cell homeostasis. For this we will measure the time dependent changes of intracellular Ca2+ and cell volume. These properties will be measured simultaneously with cellular stress to determine causality. We will further test the role of mechanosensitive channels for passing Ca2+ using specific inhibitor, GsMTx4, and explore whether pre-administration of GsMTx4 might be used as a preventive therapy.